Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and despite remarkable progress in the treatment of ALL (cure has improved from <10 percent to >75 percent), cancer remains the leading cause of death by disease in US children between 1 and 15 years of age. Methotrexate (MTX) is one of the most widely used antileukemic agents and is a component of essentially every ALL treatment protocol worldwide. Our previous work has established the advantage of high-dose (HD) MTX over low-dose, the need for higher doses in T-lineage ALL, and mechanisms for lineage and ploidy differences. However, there is currently >100-fold range in the dosage and infusion time of MTX used to treat childhood ALL, with no consideration of the genetic determinants of treatment response. Furthermore, medical economics have prompted the use of short intravenous infusions of HDMTX (e.g., 4H), so that the treatment can be delivered in an outpatient setting, yet pre clinical studies indicate greater effects with prolonged exposure. Our current research is therefore designed to determine whether 24H is superior to 4H infusion of HDMTX, for each lineage and ploidy subtype of ALL (Aim 1), and to elucidate the genetic determinants of HDMTX intracellular disposition and effects (Aims 2-4). Aim 1 is addressed in a randomized study of 4H vs. 24H infusions of HDMTX (1 gm/m2) as initial therapy of children with newly diagnosed ALL, comparing cellular accumulation and effects of the active MTX polyglutamate metabolites in bone marrow (BM) leukemia cells and in serial ALL blasts from peripheral blood. Genome-wide assessment of gene expression in ALL cells before and after treatment is used to identify genomic determinants of HDMTX cellular disposition (Aim 2), and identify treatment induced changes in gene expression that discriminate patients who have a good response (i.e., complete inhibition of de novo purine synthesis, >60 percent decrease in ALL cells by day 3, absence of submicroscopic disease in day 19 BM, complete remission at day 42, and long-term disease free survival), from patients who have a poor response (Aim 3). Aim 4 will identify genetic polymorphisms linked to differences in genomic response of candidate genes that discriminate drug effects. Preliminary data indicate that changes in gene expression can discriminate patients with a good vs. poor response, providing new insights into mechanisms of cellular resistance and revealing potential new targets to augment current treatment.